Impact and control of protozoan parasites in maricultured fishes

Buchmann, Kurt

Published in: Parasitology

DOI: 10.1017/S003118201300005X

Publication date: 2015

Document version Early version, also known as pre-print

Citation for published version (APA): Buchmann, K. (2015). Impact and control of protozoan parasites in maricultured fishes. Parasitology, 142(special issue 01), 168-177. https://doi.org/10.1017/S003118201300005X

Download date: 27. Sep. 2021 168 Impact and control of protozoan parasites in maricultured fishes

KURT BUCHMANN* Laboratory of Aquatic Pathobiology, Section of Biomedicine, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

(Received 14 November 2012; revised 8 January 2013; accepted 8 January 2013; first published online 1 March 2013)

SUMMARY

Aquaculture, including both freshwater and marine production, has on a world scale exhibited one of the highest growth rates within animal protein production during recent decades and is expected to expand further at the same rate within the next 10 years. Control of diseases is one of the most prominent challenges if this production goal is to be reached. Apart from viral, bacterial, fungal and metazoan infections it has been documented that protozoan parasites affect health and welfare and thereby production of fish in marine aquaculture. Representatives within the main protozoan groups such as amoebae, dinoflagellates, kinetoplastid flagellates, diplomonadid flagellates, apicomplexans, microsporidians and have been shown to cause severe morbidity and mortality among farmed fish. Well studied examples are Neoparamoeba perurans, Amyloodinium ocellatum, Spironucleus salmonicida, Ichthyobodo necator, Cryptobia salmositica, salmonae, Cryptocaryon irritans, Miamiensis avidus and Trichodina jadranica. The present report provides details on the parasites’ biology and impact on productivity and evaluates tools for diagnosis, control and management. Special emphasis is placed on antiprotozoan immune responses in fish and a strategy for development of vaccines is presented.

Key words: fish, parasites, protozoans, health, productivity, impact, vaccine, control.

INTRODUCTION chemical/medical intervention and immunoprophy- laxis including vaccination, which is currently used Production of teleosts in the marine environment is a for control of bacterial diseases. Metazoan parasites rapidly developing aquacultural activity worldwide. including helminths and crustaceans are also con- In relation to the known number of fish species sidered severe pests in mariculture enterprises and described, which counts more than 28000 species these parasites are in many cases visible to the naked (Nelson, 2006), relatively few of these (< 400 species) eye and therefore easily diagnosed disease agents. are currently being propagated under artificial con- Protozoans are limited in size and more difficult to ditions. However, even on this constricted basis, diagnose. If infections are observed macroscopically production of marine fish in aquaculture enterprises this will be due to pathological tissue changes is a prominent player on the world market. In 2010, (hyperplasia, hypertrophy or necrosis of host tissue) the Atlantic salmon (Salmo salar) was produced in induced by the protozoans. Despite the limited size of quantities of more than 1.5 million tonnes, milkfish protozoans their pathogenic effects on fish may be (Chanos chanos) production exceeded 0.8 million devastating and can negatively impact on fish pro- tonnes and sea bass (Dicentrarchus labrax) and sea duction. The present report provides examples of bream (Sparus aurata) rearing reached more than problems in marine fish farming caused by amoebae, 450000 tonnes (FAO, 2012). As in all other types of flagellates, apicomplexans, microsporideans and husbandry, infectious diseases represent one of the ciliates. main obstacles for safe production securing a high level of animal welfare (Rodgers and Furones, 1998; Segner et al. 2012). Viral, bacterial and fungal diseases represent well known challenges to mari- AMOEBAE culture enterprises and call for special methods for Neoparamoeba perurans successful control. Strategies rely on improved management procedures, breeding of resistant fish, Salmon farming in Tasmania, Europe, South America and North America suffers from infections with amoebae of the species Neoparamoeba perurans, * Corresponding author: Laboratory of Aquatic a parasite causing (AGD) in Pathobiology, Section of Biomedicine, Department of Atlantic salmon (S. salar) in marine fish farms Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen (Young et al. 2007; Rozas et al. 2012). It is one of 7, DK-1870 Frederiksberg C., Denmark. Tel: +45- the best documented gill diseases in salmon farming 35332700. Fax: +45-35332755. E-mail: [email protected] eliciting high morbidity and some mortality. Other

Parasitology (2015), 142, 168–177. © Cambridge University Press 2013 doi:10.1017/S003118201300005X Protozoan parasites in mariculture 169 species of Neoparamoeba have been isolated from PCR and sequencing of rDNA have been developed various marine fishes (Dyková et al. 2007) but only (Levy et al. 2007). The pathogenic effect of the para- N. perurans has been proven explicitly to elicit site is associated with severe disturbance of epithelia pathological reactions in fish gills (Young et al. which evidently can lead to osmoregulatory problems 2008). Apart from Atlantic salmon host fishes such in the host fish (Noga, 1987). Effective treatment of as coho salmon ( kisutch), chinook infections has been achieved by use of auxiliary salmon (O. tshawytscha), rainbow trout (O. mykiss), compounds containing formalin, copper and hydro- ayu (Plecoglossus altivelis), sea bass (D. labrax) and gen peroxide, but experimental trials have shown that turbot (Scophthalmus maximus) have been diagnosed also drugs such as chloride and a series of with AGD (Nowak, 2012). Amoebae induce hyper- antibiotics have effects on A. ocellatum infections in plasia in affected gill areas where inflammatory foci, fish (Noga, 2012). Epithelial sloughing and rhizoid clubbing and filament fusion may occur. Diagnosis penetration will presumably activate a series of im- requires use of molecular tools (PCR or in situ mune reactions which have not been fully described hybridization) because light microscopy cannot but the severe hyperplasia and inflammatory reac- differentiate between e.g. genera Paramoeba and tions elicited by the parasites are probably associated Neoparamoeba, both carrying eukaryotic endosym- with extensive production of cytokines. A series of bionts. Neoparamoeba perurans is a marine species antimicrobial peptides such as HLPs (histone-like and treatment can be performed by freshwater proteins) and piscidins produced in the skin of certain bathing but use of hydrogen peroxide treatments fish species show a clear killing effect on dinospores. have shown some effects as well (Nowak, 2012). It has also been shown that specific serum antibodies Irritation of gill epithelia following invasion by occur in hosts following infection and recovery amoebae lead to IL-1beta expression followed by (Smith et al. 1992; Cecchini et al. 2001) which at hyperplasia (Adams and Nowak, 2003) and higher least could partly explain the dinospore-agglutinating infections lead to extensive inflammatory responses activity of tilapia serum (Smith et al. 1993). Clown with infiltration of reactive cells. These reactions fish (Amphiprion frenatus) surviving from experi- clearly indicate that host immune responses are mental infections developed acquired immunity to activated following infection but also that they are reinfection lasting for half a year (Cobb et al. 1998) part of the pathological reactions destroying the which suggest that development of a protective architecture of the gill tissue leading to decreased vaccine may be a realistic future goal. oxygen uptake and carbon dioxide release (Nowak, 2012). Control strategies may include breeding for Ichthyodinium spp. resistant salmon strains but immunoprophylactic measures should be considered as well. Feeding The endoparasitic dinoflagellate Ichthyodinium was salmon with immunostimulating feed additives, previously known from wild fish and fish eggs but which often has been suggested to boost the fish following the advent of cod (Gadus morhua) farming immune system, did not show a satisfactory effect on (Buchmann et al. 1993; Pedersen et al. 1993) fish and infection levels (Bridle et al. 2005). Vaccines (im- eggs from these marine species were found to be infec- mersion or injection) based on live or killed amoebae ted by dinoflagellates during routine investigations and even DNA vaccines have been tested experimen- for diseases and a tentative diagnosis based on light tally but without showing any significant efficacy microscopy was made. However, a precise generic (Nowak, 2012). diagnosis was only recently obtained following sequencing of rDNA from the organism (Skovgaard et al. 2010). The parasite occurs as a trophozoite in eggs and yolk sac larvae and it was initially suggested Amyloodinium ocellatum that infection could affect host survival, a suggestion, The dinoflagellate A. ocellatum has an ectoparasitic however, that could not be confirmed by Skovgaard life style and has been considered a pest in marine et al. (2010). The absence of multiplication in eggs aquaculture for decades due to its association with and larvae of cod indicated that the stimulation severe morbidity and mortality (Paperna, 1981; of immune factors is minimal and thereby patho- Pereira et al. 2011; Saraiva et al. 2011; Soares et al. logical reactions are absent. However, Mori et al. 2012). The sessile stage on fish is termed the (2007) reported severe health and quality problems trophont, attaching itself by rhizoids penetrating associated with multiplication of Ichthyodinium surface epithelia of gills, skin and fins of host fishes. parasites in eggs and larvae of leopard coral grouper Multiplication occurs in the environment in a cyst (Plectropomus leopardus). Control efforts using stage termed the tomont. Numerous infective dino- chemical or medical treatment may be difficult due spores are released from the cyst whereafter the fish to the endoparasitic nature of this parasite and the can acquire additional infections (Noga, 1987). The fragility and vulnerability of eggs and yolk sac larvae. characteristic parasite is easily diagnosed by light Therefore it is advisable to introduce improved microscopy but molecular diagnostic tools including prophylactic hygienic measures combined with use Kurt Buchmann 170 of certified disease-free spawners, eggs and larvae in order to eliminate infections. An alternative, but still theoretical, immunoprophylactic strategy may be immunization of spawners with subsequent transfer of protective immune elements in the egg.

KINETOPLASTID FLAGELLATES Cryptobia salmositica The kinetoplastid flagellate C. salmositica infects Pacific salmon belonging to the genus Oncorhynchus and may cause considerable mortality in marine net pens. It has relatively low host specificity and may also infect sculpins (Cottus spp.) and Salvelinus fontinalis (Woo, 2012). Although leeches (Piscicola Fig. 1. Ichthyobodo necator on dorsal fin of rainbow trout. salmositica) are known to transmit these flagellates a Scale bar 5 μM. Courtesy of Dr. O. S. Møller, Zoological direct transfer from host to host under high host Museum, University of Copenhagen, Denmark. densities has been described. The parasite penetrates the fish surface and may remain in this microhabitat, but the main pathogenic effect is associated with ex- an environmental perspective, be needed in future tensive parasitaemia. The parasite’s production of production facilities should they be based on prin- cysteine- and metallo-proteases is considered to pro- ciples of sustainability. Host reactions include mu- vide the parasite with penetration tools and to be cous cell involvement and hyperplasia (Urawa, 1992; responsible for anaemia (Woo, 1979). Inflammatory Urawa et al. 1998) and recent studies have shown that reactions lead to oedema, ascites and exophthalmia experimental infections induce T cell and IgT with a general decrease in physiological functions lymphocyte depletion in skin whereas IgM-positive associated with lethargy and anorexia. The drug iso- lymphocytes accumulate in the infected epidermis metamidium chloride (Samorin), which is a known (Chettri et al. 2012). Cytokine profiling indicated that anti-trypanosomal compound, also exerts action the parasites initiate a TH2 –like response in rainbow against crytobiosis in certain stages but not the trout skin being based on up-regulation of IL-4/13 acute phase of the disease (Ardelli and Woo, 1999). and IL-10 genes. Furthermore, increased occurrence The occurrence of the flagellate in the host vascular of IgM-producing lymphocytes in the skin supports system evidently stimulates both innate and adaptive this notion. Given the recent information that rain- immune responses. High antibody titres have been bow trout utilize IgT responses combined with T cell measured in fish surviving an infection. The alterna- involvement in gills when mounting effective re- tive complement activation pathway may confer pro- sponses against white spot disease (Jørgensen et al. tection to naturally resistant fish. Several strategies 2011; Olsen et al. 2011) it may be worth reflecting on for development of vaccines against cryptobiosis have the possibility that manipulation of host responses been tested including vaccination with cultured atten- towards I. necator in a similar way could increase uated parasites (Woo and Li, 1990), recombinant pro- protection from a low to an acceptable level. tein vaccines and DNA-vaccines (Tan et al. 2008).

DIPLOMONADID FLAGELLATES Ichthyobodo necator Spironucleus salmonis was originally described as This flagellate (Fig. 1) is found both in freshwater Hexamita salmonis; it is a common intestinal parasite and marine facilities. There is debate whether more in farmed rainbow trout (Buchmann et al. 1995) eli- than one species is involved and recent studies have citing intestinal dysfunction and weight loss probably provided molecular clues to differentiation of differ- due to malabsorption (Uldal and Buchmann, 1996). ent strains and species within the genus Ichthyobodo Reports on extraintestinal occurrence of a diplo- (Isaksen et al. 2012). The parasite infects both skin monadid, previously diagnosed as H. salmonis and gill epithelia and is associated with considerable (Kent et al. 1992; Poppe et al. 1992) in maricultured morbidity and even mortalities (Urawa et al. 1998). salmonids framed the pathological importance of The parasite occurs in two forms, a motile and a diplomonadids. It was later suggested that the causa- sessile stage. The latter is probably the most tive agent was a corresponding species Spironucleus pathogenic due to its attachment and penetration of barkhanus previously recorded in grayling host epithelia. Both stages are susceptible to auxiliary (Thymallus thymallus) and arctic charr (Salvelinus chemical compounds such as formalin and various alpinus) (Sterud et al. 1998) which was suspected in oxidizing agents (hydrogen peroxide, sodium per- the spread of the infection to Atlantic salmon and in carbonate, peracetic acid). The latter group will, from the subsequent appearance of extra-intestinal Protozoan parasites in mariculture 171 abcesses. However, molecular studies showed that but also Pleistophora anguillarum in Japanese eels the pathogen was a closely related but new species, (Anguilla japonica) (Hung et al. 1996) and Entero- Spironucleus salmonicida (Jørgensen and Sterud, cytozoon spp. in Atlantic halibut (Hippoglossus 2006). The parasite’s pathogenicity is associated hippoglossus) (Bergh et al. 2001) have been associated with penetration of various tissues including muscles with pathological reactions in farmed fishes. and it has obviously an ability to evade innate re- sponses of salmon. Treatment of spironucleosis was previously conducted by the use of nitro-imidazoles Loma salmonae such as metronidazole, but due to the ban of this drug Pacific salmon cultured in marine net-pens are par- in husbandry, including fish farming, alternative ticularly affected by L. salmonae which primarily compounds have been tested for possible antiparasi- infects gill tissue but may be found in other organs tic effects. Garlic extracts were indeed found to affect (e.g. intestine) as well. Parasite spores develop in a the related flagellate Spironucleus vortens (Millet et al. host cell producing relatively large whitish xenomas 2011) which suggests that alternative drugs should be which rupture within weeks whereupon spores are tested also for S. salmonicida. Possible immuno- released to the environment. Implementation of hy- prophylactic measures including immunostimulants gienic procedures including fallowing of production and/or vaccination should be investigated. This area sites for extended time periods may be an important has many research opportunities due to the fact that prophylactic strategy. Partial treatment success the nature of the host immune response towards these against the parasitosis using fumagillin, albendazole diplomonadid flagellates has not yet been established. or monensin has been reported (Speare and Lovy, 2012). The inflammatory response to devel- opment is absent or minimal and restricted to a weak fibroblast migration towards the infected cell surface A number of apicomplexan species are common in (Speare and Lovy, 2012). This may indicate that the wild fish from both freshwater and marine habitats. parasite in this stage has an immune modulating However, reports on apicomplexan infections in mechanism securing fulfilment of the life cycle. maricultured fish are few. When Heuch et al. (2011) However, following rupture and release of spores a compared parasite infections in wild and mari- marked inflammatory response develops possibly due cultured cod (G. morhua) in Norway they detected to stimulation of damaged and exposed gill tissue by Goussia spraguei as the only species within this group external biotic and abiotic factors. The immune and occurring only in wild fish. However, turbot response following spore release is initially based on (S. maximus) farms in North West Spain were diag- neutrophil colonization followed by macrophage and nosed with scophthalmi (Alvarez- lymphocyte attraction. Even dendritic-like cells may Pellitero et al. 2004). Although no evidence for severe take part in the host response (Lovy et al. 2006). impact of infection was recorded, the authors showed Humoral host responses against microsporidian histopathological changes of the host intestinal parasites are known to occur. Japanese eel (Anguilla epithelium with leukocyte infiltration. Tissue japonica) is able to mount an antibody reaction changes associated with Cryptosporidium spp. infec- against a series of antigens in P. anguillarum tions mainly of juvenile farmed turbot were reported (Buchmann et al. 1992; Hung et al. 1996) and the from Portugal (Saraiva et al. 2009) where the parasite combined cellular and humoral reactions may be de- was found in bile duct epithelial cells. No ex- cisive elements when developing future immuno- perimental treatments of these infections were prophylactic strategies. Feeding of hosts with performed but it may be worthwhile testing if anti- immunostimulants such as beta-glucans has been de- coccidian compounds such as toltrazuril may have a scribed to diminish the infection severity (Guselle curative effect (Schmahl et al. 1989). The trend for a et al. 2010) but more effective methods should lower prevalence and intensity with older fish suggest be considered. Fish surviving an infection achieve development of age-dependent immunity against protection against reinfection which indicates these apicomplexans corresponding to anti-coccidian that vaccine development may be a realistic goal. responses in birds and mammals. This suggests that Experimental vaccines have indeed been found to further investigations on immune reactions in marine protect rainbow trout (Sánchez et al. 2001). piscine hosts should be performed in order to elucidate immune mechanisms and potentials for immunoprophylactic strategies. CILIATES A suite of ciliates has been associated with disease and mortality in mariculture. Marine white spot A range of microsporidians has been detected in disease caused by Cryptocaryon has been in focus due various maricultured fishes. One of these is Loma to its common occurrence in exhibition aquaria and salmonae in salmonids (Speare and Lovy, 2012), traditional warm water fish farming. During the last Kurt Buchmann 172

reported to be disease agents. This was partly based on recent developments within molecular biology which have improved diagnostic techniques. The small subunit ribosomal RNA gene (SSU rRNA) has proven useful for differentiation of genera, species and strains (Song et al. 2009). As a consequence, a number of other valid species within the genera Uronema and Pseudocohnilembus have been reported as pathogens in fish. Miamiensis avidus is a histo- phagous parasite invading skin, gills, muscles, brain, spinal cord and gut. The parasites are facultatively parasitic and may survive and propagate in the environment which may be problematic when control measures have been implemented. Imple- mentation of hygienic measures is the first step to Fig. 2. Invasive scuticociliate isolated from brain of prevent infection. Auxiliary substances, including turbot. Scale bar 10 μm. Courtesy of Dr J. Bresciani, formalin and hydrogen peroxide-containing com- University of Copenhagen, Denmark. pounds, are known to kill free-living stages but tissue-dwelling stages need medication that interferes with essential physiological pathways of the parasite two decades also scuticociliates have been shown to (Iglesias et al. 2002). Feeding fish with immunosti- be aggressive invaders of host fish tissues whereas mulants can induce a general immune response trichodinids only occasionally cause severe problems. which may have a controlling effect on scuticociliate infections. Among several reports Lee and Kim Cryptocaryon irritans (2009) also showed that oligonucleotides (CpG- ODN) induced resistance in olive flounder against Cryptocaryon irritans is an endoparasite penetrating M. avidus. The infected fish raise antibody responses the host epidermis, feeding on sloughed cells indu- against the ciliates and several attempts to produce a cing a marked proliferation of epidermal cells where- protective vaccine have been made. Recently high by the parasite location become macroscopically protection combined with few side-effects was visible as white spots (Yambot et al. 2003). This recorded in turbot following immunization with parasitic has a resemblance to the freshwater surface antigens of M. avidus mixed with poly- fi parasite Ichthyophthirius multi liis and has a corre- meric microspheres (León-Rodríguez et al. 2012). sponding but marine life cycle. However, analyses of Although high hygienic standards and chemotherapy rDNA sequences suggest that the two ciliates are may be convenient control measures immunopro- merely distantly related and the biological similarities phylactic methods may also have a future as can be may be due to convergent evolution (Colorni and deduced from these recent vaccine studies. Diamant, 1993). As is known from its freshwater counterpart C. irritans also induces specific antibody production in infected fish host (Misumi et al. 2011) Trichodina jadranica which suggests that immunoprophylactic measures Trichodinids (Fig. 3) are prevalent parasites both in including vaccination may be a future way of con- freshwater and marine fish culture systems. trolling the disease. Treatments using various drugs Numerous species occur in most geographic regions and chemicals (of which some are environmentally and climatic zones from arctic cod culture (Heuch friendly) have been and will probably in the near et al. 2011) to tropical bass (Lates calcarifer) pro- future be essential ways of reducing infection levels in duction (Rückert et al. 2008). A well-studied species farms (Picón-Camacho et al. 2011). is T. jadranica which is an ectoparasite with limited pathological effects on the host if the infection level is limited. However, it has the potential to harm its host Scuticociliates severely when the parasite load reaches high levels. Farmed flatfishes, such as turbot (S. maximus) and European eels (Anguilla anguilla) farming in recircu- olive flounder (Paralichthys olivaceus) and percids, lated water, both fresh and marine, have suffered such as sea bass (D. labrax), have been found invaded from recurrent attacks by T. jadranica (Madsen et al. by aggressive and invasive minute ciliates (Fig. 2). 2000a). The ciliates attach to skin, fins and gills of the Specific diagnosis of these pathogens is often European eel but do not penetrate the epidermis. hampered due to a high degree of morphological With its basic disc, reinforced by a rigid cytoskeleton similarity between these ciliates. Recent work has with contractile elements, the parasite can attach synonymized two species, Miamiensis avidus and firmly to the host surface and compress the epithelial Philasterides dicentrarchi, which have been repeatedly cell lining, whereby normal physiological functions Protozoan parasites in mariculture 173

information of a much larger part of the genome of protozoan parasites and thereby create highly precise diagnoses. Although morphometric and molecular information is basic and indispensable for the diag- nosis it should also be considered to include in vitro or in vivo infection experiments in certain problematic cases. Even small and not easily detectable mutations in a pathogen can produce very large differences in infectivity with regard to a certain host organism. Thus, a single transition in the ITS region of the helminth (Jørgensen et al. 2007) is associated with profound differences in infectivity for Atlantic salmon. Likewise, a few substitutions in the genome of the bacterium Yersinia ruckeri have created a pathogen with different antigenic properties leaving old vaccines ineffective (Desmukh et al. 2012).

Fig. 3. Trichodina sp. on caudal fin of rainbow trout. Scale bar 20 μm. Courtesy of Dr J. Bresciani, University Future disease control of Copenhagen, Denmark. Chemical control. Many diseases in fish are caused by ectoparasitic protozoans invading skin, fins and (e.g. gas exchange, ammonia release) are inhibited. gills. These organisms apply free-living life cycle Lethargy, anorexia and death may result from heavy stages (cysts, swarmers) which makes it possible infections. Although weaker individuals in the host to control the infections through water disinfection population seem to be the most susceptible no reports by use of substances such as copper sulphate and on acquired immunity are available. The ciliates feed formalin. Due to the environmentally problematic on bacteria and organic particles which explains exces- effects of these compounds recent studies have recom- sive parasite propagation in polluted water. Thus, mended alternative uses of oxidizers such as hy- it is known that mass occurrence of the parasite is drogen peroxide and related compounds (sodium connected to a high organic content of the fish farm percarbonate, peracetic acid). water (Madsen et al. 2000b). Reducing the organic load of fish farm water by mechanical filtration is therefore a relevant management tool for prevention Medical control. Several drugs with antiprotozoan ff of infections but in acute cases auxiliary chemical e ects have been developed and are available for treat- compounds such as formalin have been used to ment of humans and domestic animals. However, reduce infection levels. Due to the environmental and although experimental work has demonstrated fi health hazards connected with the use of formalin marked activity against sh protozoans, their use alternative compounds are recommended for control. within aquacultural enterprises is limited due to Oxidizers including hydrogen peroxide-containing lack of licensing and to legislative restrictions. products and even garlic juice were found to be Nitroimidazoles such as metronidazole and dimetri- ff fl effective for elimination of these parasites (Madsen dazole show a strong e ect on agellates and ciliates et al. 2000a). and this drug group is allowed for human use but not for food production animals. Likewise, the anti- coccidian drug toltrazuril can be used for prevention of ciliate infections in fish (Jaafar and Buchmann, DISCUSSION 2011). It is licensed for use in the poultry and pig Future diagnostic methods industry but it is questionable if the drug can be licensed for aquacultural animals as well. Early classical descriptions of protozoans have relied on line drawings and light microscopy photo- graphs with written reports on morphometric obser- Management. Strict hygienic measures, quarantine vations. Later, scanning and transmission electron practices, compulsory use of certified disease-free microscopy improved the diagnostic precision (Lom fish, fallowing of production sites and elimination of and Dyková, 1992) and during recent decades se- infective parasite stages by various filtration tech- quencing of selected gene regions such as rDNA and niques are practices which should be used in any mitochondrial DNA have improved resolution even production system. Thus, water filtration by the use further. It is to be expected that development of of mechanical filters may (dependent on the mesh sequencing techniques will allow us to obtain size) remove potentially harmful tomonts of Kurt Buchmann 174

C. irritans and thereby prevent multiplication within Vaccination. Disease control in aquacultural enter- tomocysts. prises must ideally be based on integrated systems applying high hygienic standards. This can be Breeding of innately resistant fish. Mechanisms achieved through filtration of water (with mechanical involved in and responsible for innate resistance and biological filters) securing optimal physico- of specified stocks and species of fish towards chemical water parameters, by breeding for resistant pathogens have not been fully elucidated. However, fish strains and feeding at an optimal level. However, a range of examples is available. A well-documented additional control methods must be considered example is the innate resistance of several Baltic in order to avoid medication. Vaccination is an salmon strains against the ectoparasitic monogenean immuno-prophylactic measure which can provide G. salaris (Bakke et al. 1990; Dalgaard et al. 2003; the fish with a high resistance against infection. Lindenstrøm et al. 2006). In addition, susceptibility Significant results with development of antibacterial and resistance of various salmonids towards the vaccines have been achieved in the Norwegian salmon bacterial pathogen was also industry where the use of antibiotics has been reduced reported to vary significantly between salmon strains by 99 % during the latest 26 years despite a 20-fold (Holten-Andersen et al. 2012). It is therefore relevant increase of the annual salmon production. High to initiate breeding programmes in order to develop efficacies of anti-viral vaccines have also been demon- strains with a low susceptibility towards pathogens strated (Lorenzen et al. 2000) and efforts should including protozoans. therefore be made in order to develop antiparasitic vaccines. Due to the limited size of many protozoan Immunoprophylaxis. The potential and capacity parasites the cellular and humoral immunological of the teleost immune system can be appreciated armament in fish should have the basic ability to through recent discoveries which have suggested that develop protective immunity against at least some immunoprophylactic measures may be significant of these pathogens. Experimental vaccines against ways to secure fish health. Some strategies in the field C. salmositica (Woo, 2012), L. salmonae (Sánchez et al. are based on feeding of fish with compounds often 2001; Rodríguez-Tovar et al. 2006) and I. multifiliis termed immunostimulants which are chemicals (the freshwater equivalent to C. irritans) (Alishahi and stimulating innate immune parameters. Other strat- Buchmann, 2006) have been found to be partially egies apply specific immunization of their production effective which is promising for further achievements. animals against pathogens. Cryptocaryon irritans is known to elicit antibody production in affected fish (Misumi et al. 2011) which Immunostimulants. A series of structural molecules is a valuable basis for further vaccine development. from various sources e.g. bacteria, yeast, fungi Experimental studies have also demonstrated sig- and lichens alone or together with synthetic products nificant activation of cellular and humoral immune (e.g. oligonucleotides) is currently used as im- factors in fish exposed to several other protozoans munostimulants. A large group of compounds on such as A. ocellatum (Noga, 2012) and I. necator this product shelf is the beta-glucans (Skov et al. (Chettri et al. 2012). Thus, it is likely that production 2012). The immunostimulants possess pathogen- of vaccines against A. ocellatum, S. salmonicida, associated molecular patterns (PAMPS) which are I. necator and L. salmonae may be realistic goals for able to stimulate different pathogen recognition re- renewed research efforts. However, it cannot be ceptors (PRRs) in host fishes and thereby activate excluded that the parasites themselves or parasite- innate immune reactions (Chettri et al. 2011). These derived molecules (Jørgensen and Buchmann, 2011) comprise among others toll-like receptors which divert the response of the host towards a less effective subsequently can initiate cascade reactions which immune pathway and this possibility should be taken may limit or reduce infections. Results from con- into account when designing vaccines and their trolled laboratory (Jaafar et al. 2011)orfield studies adjuvants. The recent development of reagents and (Xueqin et al. 2012) have indicated that immune tools for investigating immune mechanisms in fish can elements are activated by these feed additives. create a basis for tailor-made vaccines stimulating Unfortunately the effect of these feed additives immune mechanisms in fish hosts which can provide on parasite infection levels, such as intensities of protection. the skin parasitizing ciliate I. multifiliis, is limited. Correspondingly, immunostimulant feeding does CONCLUSIONS AND FUTURE DIRECTIONS not result in a clear reduction of L. salmonae infection in salmonids but merely leads to a more benign Control of protozoan infections of maricultured course of infection (Guselle et al. 2010). Likewise, fishes must be performed through integrated efforts AGD in salmon is not diminished by in-feed im- applying prophylactic management methods creating munostimulants (Bridle et al. 2005). Therefore other a better physical environment for the captive fishes. immunoprophylactic strategies are needed in order to Breeding for natural resistance and improved nutri- secure a satisfactory fish health level. tion should also be a priority area which can add to a Protozoan parasites in mariculture 175 high health status in mariculture enterprises. Alvarez-Pellitero, P., Quiroga, M. I., Sitja-Bobadilla, A., Redondo, M. J., Palenzuela, O., Padros, F., Vazquez, S. and Application of chemicals and drugs may be necessary Nieto, J. M. (2004). Cryptosporidium scophthalmi n. sp. (Apicomplexa: tools during acute outbreaks but more sustainable Cryptosporidiidae) from cultured turbot Scophthalmus maximus. Light and methods should be implemented to avoid infections. electron microscope description and histopathological study. Diseases of Aquatic Organisms 62, 133–145. It should be noted that vaccine development is a Ardelli, B. F. and Woo, P. T. K. (1999). The therapeutic use of central task to be performed in order to exploit the isometamidium chloride against Cryptobia salmositica in rainbow trout high potential of the fish immune system and reach (Oncorhynchus mykiss). Diseases of Aquatic Organisms 37, 195–203. fi Bakke, T. A., Jansen, P. A. and Hansen, L. P. (1990). Differences in sustainability in sh farming. At present, most host resistance of Atlantic salmon Salmo salar L. stocks to the monogenean experimental approaches can be considered rather Gyrodactylus salaris Malmberg, 1957. Journal of Fish Biology 37, primitive and coarse vaccines based on crude parasite 577–587. ffi Bergh, O., Nilsen, F. and Samuelsen, O. B. (2001). Diseases, prophylaxis preparations may not be su cient for obtaining and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review. protection and reaching production goals and high Diseases of Aquatic Organisms 48,57–74. welfare of farmed fish. Basic research on immune Bridle, A. R., Carter, C. G., Morrison, R. N. and Nowak, B. F. (2005). fi The effects of beta-glucan administration on macrophage respiratory mechanisms responsible for protection of sh against burst activity in Atlantic salmon challenged with amoebic gill disease protozoan diseases can direct future production of (AGD) – evidence of inherent resistance. Journal of Fish Diseases 28, tailor-made vaccines for specific parasitic diseases 347–356. Buchmann, K., Dalsgaard, I. and Larsen, J. L. (1993). Diseases and (Jørgensen and Buchmann, 2011). The immunologi- injuries associated with mortality of hatchery reared Baltic cod (Gadus cal network in fish is extensive and different parasites morhua L.) larvae. Acta Veterinaria Scandinavica 34, 385–390. may lead the immune reactions in directions which Buchmann, K., Lyholt, H. K. and Uldal, A. (1995). Parasite infections in Danish trout farms. Acta Veterinaria Scandinavica 36, 283–298. are not optimal for protection. It has been shown that Buchmann, K., Ogawa, K. and Lo, C.-F. (1992). Immune response of the development of reagents and their use in a range the Japanese eel (Anguilla japonica) against major antigens from the of assays such as immunohistochemistry, immuno- microsporean Pleistophora anguillarum) Hoshina, 1951. Fish Pathology 27, fl 157–161. cytochemistry, ow-cytometry, gene expression Buchmann, K. and Pedersen, K. (1994). A study on teleost phylogeny technology combined with in vivo challenge studies using specific antisera. Journal of Fish Biology 45, 901–903. can improve our understanding of fish immune Cecchini, S., Saroglia, M., Terova, G. and Albanesi, F. (2001). Detection of antibody response against Amyloodinium ocellatum (Brown, reactions, their diversity and potential. Due to 1931) in serum of naturally infected European sea bass by an ezyme-linked limited cross-reactivity of reagents developed for immunoabsorbent assay (ELISA). Bulletin of the European Association for salmonids (e.g. rabbit antibodies reacting with fish Fish Pathologists 21, 104–108. Chettri, J. K., Holten-Andersen, L., Raida, M. K., Kania, P. and IgM) with regard to cyprinids, anguillids, percids, Buchmann, K. (2011) PAMP induced expression of immune relevant gadids and pleuronectids (Buchmann and Pedersen, genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss). 1994) it is necessary to initiate these research Developmental and Comparative Immunology 35, 476–482. fi fi Chettri, J. K., Kuhn, J. A., Jaafar, R. M., Kania, P. W., Møller, O. S. and initiatives for all the main sh taxa. Speci c reagents Buchmann, K. (2012). Immune response of rainbow trout juveniles to the for the major fish groups in aquaculture should be protozoan parasite Ichthyobodo necator: immunohistochemical and gene developed in order to provide a basis for elucidation expression studies. In Immune Responses in Fish. DAFINET Workshop (ed. fi Kania, P. W. and Buchmann, K.), p. 6. Frederiksberg Bookprinter, of sh immune responses against protozoans in Frederiksberg, Denmark (www.dafinet.dk). mariculture. Priorities should be reagents to describe Cobb, C. S., Levy, M. G. and Noga, E. J. (1998). Development of innate, adaptive responses, cellular (macrophage, T immunity by the tomato clownfish Amphiprion frenatus to the dinoflagellate parasite Amyloodinium ocellatum. Journal of Aquatic Animal Health 10, cell and B cell markers) and humoral elements 259–263. (various immunoglobulin classes, complement fac- Colorni, A. and Diamant, A. (1993). Ultrastructural features of tors, acute phase reactants, cytokines) within these Cryptocaryon irritans, a ciliate parasite of marine fish. European Journal of Protistology 29, 425–434. reaction pathways. This basic knowledge may even- Dalgaard, M. B., Nielsen, C. V. and Buchmann, K. (2003). Comparative tually lead to optimization of vaccines not only susceptibility of two races of Salmo salar (Baltic Lule river and Atlantic for fish in mariculture but also for higher vertebrates. Conon river strains) to infection with Gyrodactylus salaris. Diseases of Aquatic Organisms 53, 173–176. Desmukh, S., Raida, M. K., Dalsgaard, I., Chettri, J. K., Kania, P. W. and Buchmann, K. (2012). Comparative protection of two different ACKNOWLEDGEMENTS commercial vaccines against Yersinia ruckeri serotype O1 and biotype 2 in rainbow trout (Oncorhynchus mykiss). Veterinary Immunology and This work was produced under the Danish Fish Immunopathology 145, 379–385. Immunology Research Centre and Network DAFINET Dyková, I., Nowak, B. F., Peckova, H., Fiala, I., Crosbie, P. and (www.dafinet.dk) supported by the Danish Council for Dvorakova, H. (2007). Phylogeny of Neoparamoeba strains isolated from Strategic Research. marine fish and invertebrates as inferred from SSU rDNA sequences. 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